1. Field of the Invention
The present invention relates to a method of assaying or analyzing subtypes of interferons derived from human leukocytes and antibodies against the said subtype, and to materials being usable for the method.
2. Description of the Related Art
Human leukocyte interferon, also called human interferon alpha, is known to exhibit antiviral and anti-tumor activities, and its clinical application has already been started. The recently commercialized genetic engineering has quickly been applied for the product and prompted greatly the analyses of the genes for interferons. The human interferons are classified into three types, or alpha, beta and gamma, according to the differences in kind of the cells from which they are derived and their physico-chemical properties. With reference to the beta-interferon, two molecular species have been reported to exist, while one molecular species has been described for the gamma-interferon. In contrast to these interferons, the alpha-type of interferon is known to exist as a great variety of subtype as many as more than 20 kinds (Goeddel et al., Nature, 287, 411 (1980). Pestka et al., 1987 Annual Review of Biochemistry, 56, 727), and extensive investigation has been carried out into the commercialization of the said interferon. As an analysis for such interferons, however, there has been used a extremely complicated technique which comprises purifying each of the subtypes to a high degree of purity, followed by amino acid sequencing (Rubinstein et al., Proc. Natl. Acad. Sci. USA, 76, 640 (1979)). The individual subtype, although containing a particular amino acid sequence in common, differ each from the other partially in amino acid sequence and have been clarified to be mutually independent gene expressions. In exerting the activities, they offer different specific features in species-specificity spectrum of antiviral activity, etc., and are considered to share physiological activities or cooperate among each other in some form in living body. Nevertheless it is quite difficult to except that such subtypes allow all of their genes to undergo expression in living body, and though the protein sequencing analysis of the subtypes presents an important problem in the analysis of their activities, the lack of a simple and convenient analytical technique has made the range of findings extremely restricted and limited in this field (Adolf et al., J. Gen. Virol., 68, 1669 (1987)). After recombinant alpha-type interferon produced by use of genetic engineering has been initiated to be applied to humans, in addition, the production of neutralizing antibody against interferons is in recent years observed at high frequencies.
In view of the fact that recombinant interferon is composed in nature of a single subtype, the incidence of such a kind of antibodies is assumed to nullify the effect of the administered interferons, and can be considered the critically serious problem from the viewpoint of clinical application. Also, such a kind of antibodies is thought to neutralize the activities of the interferons that the human body produces as a part of its own defense mechanism, and is presenting an entirely novel category of problem. Yet, the lack of an effective evaluation procedure of antibodies has resulted in failure to accumulate relevant findings. As is stated above, the development of a simple, convenient and quick analyzing method for the subtype compositions and antibodies is in strong demand from the standpoints of fundamental science and clinical application. For the purpose of analyzing all the naturally occurring subtypes, it is necessary to prepare the antibody using as an antigen the partially purified interferons containing all subtypes. In preparing the antibody, it is preferable to immunize two species of animals in order to establish an analysis technique based on the sandwich type enzyme-antibody assay through the two-antibody method, but it may also be possible to prepare the antibody with use of one species of animal, followed by direct enzyme-labelling of part of the antibody to perform sandwiching. In either case, the resulting antibody must be a polyclonal antibody capable of recognizing all the subtypes. For the purpose of this, it may be possible to use a suitable mixture of monoclonal antibodies against individual subtype, but it is difficult to establish the hybridomas which can produce such antibodies. This is because this technique suffers the defects that (1) all the subtypes of interferons must be isolated and purified in fairly large quantities, (2) individual cell fusions must be effected, (3) the cells capable of producing the antibodies must be screened and furthermore the resulting antibodies can only recognize one antibody-determining portion out of the protein of each subtype, leading to a very fair possibility of escaping detection in the case of subtype produced only in very minute quantities. In the case of a polyclonal antibody against leukocyte interferon, contamination with antibodies against impurities contained in the purified sample is likely to take place, and this must be absolutely avoided.